Current treatments for many types of cancer are primitive. Chemotherapies and radiation treatments kill diseased and healthy cells indiscriminately resulting in great discomfort to the patient and many side-effects. Understanding the molecular events involved in tumorigenesis will enable the development of cancer therapies that target specific genes and proteins within diseased cells producing therapies that are more effective and less toxic to the patient. The development of a method for rapidly screening potential therapies and molecular targets would greatly enhance the ability of researchers to develop new targeted cancer treatments that are less toxic to healthy cells.
The entire sequence of the human genome is now published, however the function of many genes remain unassigned. Recent advances in DNA microarray technology have allowed for the identification of genes expressed in many human cell types and provided a method of detecting abnormal gene expression in cancer cells. Hundreds of genes whose expression is altered in cancer cells have been identified in this way. Current functional genomic technologies are limited since only one gene can be analyzed at a time, and these techniques are unable to determine the extent to which each gene contributes to abnormal growth in cancer cells when multiple genes are involved.
Diseased cells may contain a mutated or mis-expressed gene that effects several protein signaling pathways that each contribute to the cancerous phenotype. A particularly effective cancer treatment would either specifically target the affected gene or target genes in each of the affected pathways. In either case, it is necessary to understand the expression patterns of a great number of potential targets and to have methods for modulating expression of each potential target. Thus, a high-throughput method for functional genomic screening and inhibitor design is required for the identification of cancer therapeutic agents.
Anchorage-independent growth is the gold-standard for in vitro testing of potential chemotherapeutic agents. Tumorigenic cells do not exhibit contact inhibition and can grow independently of extracellular matrix (ECM) binding. Normal cells cannot. Therefore, only tumorigenic cells grow in three dimensions on soft agar devoid of ECM. Currently, anchorage-independent growth assays can only test one inhibitor at a time or several inhibitors in 96 well plates. A separate transfection step is required in either case, and these methods require a large amount of biopsy tissue to obtain sufficient colony growth and cells need to be stained for quantification. A high-throughput assay for anchorage-independent growth that does not require killing the cells prior to imaging is desirable. In addition, one-step transfection is desirable.
The limitations described above and the ability of tumorigenic cells to grow in the absence of ECM make it desirable to develop a reverse transfection microarray system in which cells are grown in a matrix devoid of ECM. This system will use less biopsy tissue and allow for tumorigenic cell specific growth while more closely mimicking the in vivo environment of cancer cells.